Melanoma-inhibiting protein

ABSTRACT

The invention concerns a melanoma-inhibiting protein, nucleic acid sequences coding for this protein, process for the isolation of this protein as well as its use for the production of a therapeutic agent.

FIELD OF THE INVENTION

The invention concerns a melanoma-inhibiting protein (MIA), a nucleicacid which codes for it, a process for the isolation and for thedetection of this protein as well as its use for the production of atherapeutic agent.

BACKGROUND AND PRIOR ART

The regulation of cell growth is controlled by factors which actpositively as well as negatively. The factors with a positive effectinclude the known growth factors such as e.g. epidermal growth factor(EGF), platelet derived growth factor (PDGF), insulin and somatomedins.The factors with a negative i.e. inhibitory activity include, inaddition to TGF-β which can act as a growth stimulator as well as agrowth inhibitor (Roberts et al., Proc. Natl. Acad. Sci. 82 (1985),119-123), the endogenous, tumour-inhibiting factors from colon carcinomacells (Levine et al., Cancer Research 45 (1985), 2248-2254), melanomas(Bogdahn et al., Cancer Research 49 (1989), 5358-5363) as well as fromhealthy epithelial cells from the mammary glands of the rat (Ethier etal., J. Cell. Phys. 142 (1990), 15-20).

Disturbances of this regulatory system such as for example byoverproduction of growth factors with a positive action or by a reduceddependence of mutated cells on these growth factors (Rodeck et al.,International Journal of Cancer 40 (1987), 687-690) enable tumour cellsto proliferate in an uncontrolled manner. The aforementionedtumour-inhibitory factors from various tumour tissues representinteresting compounds which may be able to intervene therapeutically inthis impaired regulatory system. It must be possible to provide thesefactors in large amounts and in reproducible purity for such atherapeutic use. However, for most of these factors only enrichedfractions from cell lysates have been described up to now which are notsuitable for a therapeutic application due to their complex andsometimes unknown composition and the concomitant non-reproducibility oftheir production.

SUMMARY OF THE INVENTION

The invention is based on a new melanoma-inhibiting protein (denoted MIAprotein, or MIA, in the following) which inhibits growth of the celllines HTZ 19-dM and ATCC CRL 1424 and

a) is coded by the DNA sequence shown in SEQ ID NO: 1 for the matureprotein or for the protein with a N-terminal pre sequence, or by thegenomic sequence shown in SEQ ID NO: 3,

b) is coded by DNA sequences which hybridize with the DNA sequencesshown in SEQ ID NO: 1 or 3 or fragments of these DNA sequences in theDNA region which codes for the mature protein.

Growth inhibition is to be understood as an anti-proliferative activity.In this process the growth of the cells is considerably retarded byaddition of the MIA protein to the culture medium. A suitableconcentration for this is for example 0.1 μg MIA protein/ml culturemedium. Higher or lower concentrations of the MIA protein are, however,also suitable for growth inhibition which is observed to be higher orlower depending on the concentration.

The properties of such a protein are described by the inventors inCancer Research 49 (1989), 5358-5363, Cancer Research 50 (1990),6981-6986, Melanoma Research 2 (1992), 327-336. However, a process forthe production of this protein which can be reproduced is not stated inthese publications. The protein is obtainable from the human melanomacell line HTZ 19-dM which was previously not yet available to thepublic. This cell line was derived from a metastizing malignant melanomaand cultured as a monolayer culture in a defined serum-free culturemedium (50% Dulbecco's minimal essential medium, 50% F-12) containing0.8 mmol/l L-glutamine, non-essential amino acids, 10 μg/ml transferrin,30 nmol/l sodium selenite and 4 μg/ml gentamicin under standard cultureconditions. The cell line was deposited at the "Deutsche Sammlung furMikroorganismen und Zellkulturen GmbH" in Braunschweig on the 23.06.93(DSM ACC 2133). It is also a further subject matter of the invention.The protein according to the invention can be obtained from the culturesupernatant of this cell line by gel chromatographic isolation of aprotein fraction having a size of ca. 11 kD and subsequent purificationof this fraction by means of reverse phase HPLC.

The protein can be defined by its DNA sequence and by the amino acidsequence derived therefrom. The MIA protein can occur in natural allelicvariations which differ from individual to individual (e.g. SEQ ID NO:24). Such variations of the amino acids are usually amino acidsubstitutions. However, they may also be deletions, insertions oradditions of amino acids to the total sequence. The MIA proteinaccording to the invention--depending, both in respect of the extent andtype, on the cell and cell type in which it is expressed--can be inglycosylated or non-glycosylated form.

The protein according to the invention can also be produced byrecombinant means. Non-glycosylated MIA protein is obtained when it isproduced recombinantly in prokaryotes. With the aid of the nucleic acidsequences provided by the invention it is possible to search for the MIAgene or its variants in genomes of any desired cells (e.g. apart fromhuman cells, also in cells of other mammals), to identify these and toisolate the desired gene coding for the MIA protein. Such processes andsuitable hybridization conditions are known to a person skilled in theart and are described for example by J. Sambrook in Molecular cloning,Cold Spring Harbor Laboratory, 1989 and B. D. Hames, S. G. Higgins,Nucleic acid hybridisation--a practical approach (1985) IRL Press,Oxford, England. In this case the standard protocols described in thesepublications are usually used for the experiments.

The use of recombinant DNA technology enables the production of numerousMIA protein derivatives. Such derivatives can for example be modified inindividual or several amino acids by substitution, deletion or addition.The derivatization can for example be carried out by means of sitedirected mutagenesis. Such variations can be easily carried out by aperson skilled in the art (J. Sambrook, B. D. Hames, Loc. Lit.). Itmerely has to be ensured that the characteristic properties of the MIAprotein (inhibition of the aforementioned cell lines) is preserved.

The invention therefore in addition concerns a MIA protein which

a) is a product of a prokaryotic or eukaryotic expression of anexogenous DNA,

b) is coded by the DNA sequence shown in SEQ ID NO: 1 for the matureprotein or for the protein with an N-terminal pre sequence, the genomicsequence shown in SEQ ID NO: 3,

c) is coded by DNA sequences which hybridize with the DNA sequencesshown in SEQ ID NO: 1 or 3 or fragments of the DNA sequences in the DNAregion which codes for the mature protein, or

d) is coded by DNA sequences which if there was no degeneracy of thegenetic code, would hybridize with the sequences defined in b) to c) andcode for a polypeptide with amino acid sequence.

A protein is preferred which is coded by nucleotides 40-432 or 112 to432 from SEQ ID NO: 1, or by DNA sequences which due to genetic codedegeneracy would code for a polypeptide with the same amino acidsequence.

The MIA protein from HTZ 19-dM has a molecular weight of ca. 11 kD, isthermo-stable (3 minutes at 100° C.) and is sensitive towards proteasessuch as e.g. trypsin.

The invention concerns a nucleic acid which codes for a MIA protein andis selected from the group

a) DNA sequences shown in SEQ ID NO: 1 and 3 or the complementarysequences,

b) nucleic acid sequences which hybridize with one of the sequences froma),

c) nucleic acid sequences which, if there was no degeneracy of thegenetic code, would hybridize with one of the sequences stated in a) orb).

The invention additionally concerns melanoma-inhibiting proteins frommammalian cells, such as, e.g., mouse, rat, bovine animal, sheep, whichinhibit in an essentially analogous manner the growth of the cell linesHTZ19-dM and ATCC CRL1424, such as the human MIA protein.

These proteins which are analogous to the human MIA protein can beobtained by screening a cDNA library of the respective mammalian with ahybridization sample containing sequences coding for human MIA,according to methods familiar to the skilled artisan, carrying out asequence comparison of the DNA and the protein sequence for human andmurine MIA (SEQ ID NO: 1-5) and identifying the coding fragment.

A preferred embodiment of the invention is the murine MIA protein andthe nucleic acid sequence coding therefor (SEQ ID NO:4). The murineprotein is coded by nucleotides 110-499 or 179-499 of SEQ ID NO: 4.

With the aid of these nucleic acids the protein according to theinvention can be obtained in a reproducible manner and in large amounts.For expression in prokaryotic or eukaryotic organisms, such asprokaryotic host cells or eukaryotic host cells, the nucleic acid isintegrated into suitable expression vectors, according to methodsfamiliar to a person skilled in the art. Such an expression vectorpreferably contains a regulatable/inducible promoter. These recombinantvectors are then introduced for the expression into suitable host cellssuch as e.g. E. coli as a prokaryotic host cell or Saccharomycescerevisiae, Terato carcinoma cell line PA-1 sc 9117 (Buttner et al.,Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells, CHO or COS cells aseukaryotic host cells and the transformed or transduced host cells arecultured under conditions which allow an expression of the heterologousgene. The isolation of the protein can be carried out according to knownmethods from the host cell or from the culture supernatant of the hostcell. Such methods are described for example by Ausubel I., FrederickM., Current Protocols in Mol. Biol. (1992), John Wiley and Sons, NewYork. Also in vitro reactivation of the protein may be necessary.

A DNA with the nucleotides 40-432 or 112-432 (coding sequence) of SEQ IDNO: 1 (cDNA) or the genomic DNA according to SEQ ID NO: 3 is preferablyused for the recombinant production of the protein according to theinvention.

In addition the invention concerns a process for obtaining a MIA proteinby isolation of the culture supernatant of the melanoma cell line HTZ19-dM by means of a gel chromatographic separation and purification of afraction which corresponds to a molecular weight of ca. 11 kD (SDS-PAGE,non-reduced) by means of reverse phase HPLC. Ca. 0.2 μg/l culturesupernatant can be obtained in this way.

In a preferred embodiment, the natural MIA protein, during isolation andpurification, is subjected to acid treatment. By this, the MIA activitycan be enhanced. Advantageously, a pH value of about 2 is applied; asacid, e.g., acetic acid is suitable.

The detection of transformed or transduced host cells whichrecombinantly produce the MIA protein and the purification of theprotein are preferably carried out by means of antibodies which bind tothis protein. Such antibodies can be obtained in a simple manneraccording to known methods by using the protein according to theinvention as an antigen or an immunogen.

The invention therefore in addition concerns the use of the protein withmelanoma-inhibiting activity according to the invention for theproduction of antibodies which bind to this protein.

For this animals which are usually used for this purpose, such as inparticular, sheep, rabbits or mice, are immunized with the proteinaccording to the invention and subsequently the antiserum is isolatedfrom the immunized animals according to known methods or spleen cells ofthe immunized animals are fused with immortalized cells, such as e.g.myeloma cells, according to the method of Kohler and Milstein (Nature256 (1975), 495-497). Those cells which produce a monoclonal antibodyagainst the MIA protein are selected from the hybridoma cells obtainedin this way and cloned. The monoclonal or polyclonal antibodies obtainedin this way can be bound to a support material, such as e.g. cellulose,for an immunoabsorptive purification of the melanoma-inhibiting protein.Furthermore antibodies of this kind can be used for the detection of theMIA protein in samples, such as e.g. cut tissue or body fluids.

The invention therefore additionally concerns antibodies against the MIAprotein which are obtainable by immunizing an animal with a MIA proteinand isolating the antibodies from the serum or spleen cells of theimmunized animals.

It has turned out that the MIA protein not only exhibits an inhibitoryactivity on melanoma cells but also, to a smaller extent, on othertumour cells such as e.g. glioblastoma cells, neuroblastomas, small celllung cancer and neuroectodermal tumours by inhibiting DNA synthesis (³H-thymidine incorporation (Coligan J. E., Kruisbeek A. M., Margulies D.H., Shevach E. M., Strober W., Current Protocols Immunology, NIHMonograph, J. Wiley and Sons, New York, 1992)), inhibiting tumour colonyformation in soft agar or in a tumour stem cell assay (Schlag P.,Flentje D., Cancer Treatment Rev. 11: Suppl. A: 131-137, 1984). Incontrast the growth of normal non-degenerate cells is not inhibited.This protein acts already at very low concentrations (nanogram range).This protein is therefore suitable for the production of a therapeuticagent for tumour therapy. Such a therapeutic agent is particularlysuitable for the therapy of malignant melanomas, malignant gliomas,bronchial carcinomas (in particular small cell bronchial carcinoma,SCLC) and neuroblastomas.

It has in addition turned out that the melanoma-inhibiting proteinsuppresses the interleukin 2-dependent and phytohaemagglutinin-inducedproliferation of peripheral blood lymphocytes. The cytotoxicity of Tlymphocytes is also reduced. The melanoma-inhibiting protein is thusalso suitable for the production of a therapeutic agent which can beused as an immuno-suppressive agent.

The invention therefore in addition concerns the use of a proteinaccording to the invention for the production of a therapeutic agentwhich can be used in tumour therapy or as an immunosuppressive agent.

The protein according to the invention is processed, if desired togetherwith the usually used auxiliary agents, fillers and/or additives, in apharmaceutical formulation for the said therapeutic applications.

The invention therefore in addition concerns a therapeutic compositioncontaining a melanoma-inhibiting protein according to the invention andif desired together with the auxiliary agents, fillers and/or additivesthat are usually used.

The invention further concerns the use of sequences of the MIA gene,preferably sequences coding for a protein having MIA activity, oractivating sequences from the 5' untranslated region, in gene therapy,and in particular, for the production of medicaments for gene therapy.

Gene therapy of somatic cells can be accomplished by using, e.g.,retroviral vectors, other viral vectors, or by non-viral gene transfer(for clarity cf. T. Friedmann, Science 244 (1989) 1275; Morgan 1993, RACDATA MANAGEMENT REPORT, June 1993).

Vector systems suitable for gene therapy are, for instance, retroviruses(Mulligan, R. C. (1991) in Nobel Symposium 8: Ethiology of human diseaseat the DNA level (Lindsten, J. and Pattersun Editors), pages 143-189,Raven Press), adeno associated virus (McLughlin, J. Virol. 62 (1988),1963), vaccinia virus (Moss et al., Ann. Rev. Immunol. 5 (1987) 305),bovine papilloma virus (Rasmussen et al., Methods Enzymol. 139 (1987)642) or viruses from the group of the herpes viruses such as EpsteinBarr virus (Margolskee et al., Mol. Cell. Biol. 8 (1988) 2937) or Herpessimplex virus.

There are also known non-viral delivery systems. For this, usually"nude" nucleic acid, preferably DNA, is used, or nucleic acid togetherwith an auxiliary such as, e.g., transfer reagents (liposomes,dendromers, polylysine-transferrine-conjugates (Wagner, 1990; Felgner etal., Proc. Natl. Acad. Sci. USA 84 (1987) 7413)).

Another preferred method of gene therapy is based on homologousrecombination. In this, either the gene coding for the MIA protein canbe inserted in one or more copies into the genome of somatic cellsand/or the MIA gene endogenously present in the cells can be modulated,preferably activated.

Methods of homologous recombination are described, e.g., inKucherlapati, Proc. in Nucl. Acids Res. and Mol. Biol. 36 (1989) 301;Thomas et al., Cell 44 (1986) 419-428; Thomas and Capecchi, Cell 51(1987) 503-512; Doetschman et al., Proc. Natl. Acad. Sci. USA 85 (1988)8583-8587 and Doetschman et al., Nature 330 (1987) 576-578. In thesemethods, a portion of DNA to be ingrated at a specific site in thegenome (gene fragment of MIA) is bound to a targeting DNA. The targetingDNA is a DNA which is complementary (homologous) to a region (preferablywithin or proximal to the MIA gene) of the genomic DNA. When twohomologous portions of a single-stranded DNA (e.g. the targeting DNA andthe genomic DNA) are in close proximity to one another they willhybridize and form a double-stranded helix. Then the MIA gene fragmentand the targeting DNA can be integrated into the genome by means ofoccurrence of recombination. This homologous recombination can becarried out both in vitro and in vivo (in the patient).

Preferably, there is used a DNA which codes for a protein having MIAactivity, a fragment which inhibits MIA expression (knock-out sequence)or a fragment capable of activating, after integration of the genome ofa cell, expression, in this cell, of a protein having MIA activity. Sucha fragment may be, for example, a promoter and/or enhancer region whichis heterologous to the corresponding MIA region or which, afterintegration into the MIA gene, activates the actually silent or to alittle extent expressed MIA gene transcriptionally and/ortranslationally.

Thus, by means of this DNA, one or more MIA genes are newly introducedinto the target cell, or the essentially transcriptionally silent genein the genome of a mammalian cell is activated in such fashion that themammalian cell is enabled to produce endogenous MIA protein. To thisend, a DNA construct is inserted into the genome by homologousrecombination, the DNA construct comprising the following: a DNAregulatory element capable of modulating, preferably stimulating,expression of this gene if operatively linked thereto; and one or moreDNA target segments which are homologous to a region in this genome,which region is within or proximal to this gene. This construct isinserted into the genome of the mammalian cell in such fashion that theregulatory segment is operatively linked to the gene which codes for theprotein having MIA activity. Preferably, the construct further comprisesamplifying sequences, especially if genes coding for proteins with MIAactivity are inserted into the cell.

For the introduction of MIA genes into the target cells, the constructcomprises a regulatory element, one or more MIA genes and one or moretarget segments. The target segments are chosen in such a way that theyhybridize with an appropriate region of the genome, whereby, afterhomologous recombination, the inserted exogenous MIA genes areexpressed.

There are known a large number of processes by which homologousrecombination can be initiated. Preferably, homologous recombinationtakes place during DNA replication or mitosis of the cells. A DNA ofthis kind can be used for the production of an agent for therapeutictreatment of tumours or for the production of homologous or heterologousMIA protein in a host organism.

It is possible to provide a test on the basis of the nucleic acidsequences of the MIA protein provided by the invention which can be usedto detect nucleic acids which code for MIA proteins. Such a test can forexample be carried out in cells or cell lysates. Such a test can becarried out by means of nucleic acid diagnostics. In this case thesample to be examined is brought into contact with a probe which wouldhybridize with the nucleic acid sequence coding for the MIA protein. Ahybridization between the probe and nucleic acids from the sampleindicates the presence of expressed MIA proteins. Such methods are knownto a person skilled in the art and are for example described in WO89/06698, EP-A 0 200 362, USP 2915082, EP-A 0 063 879, EP-A 0 173 251,EP-A 0 128 018.

In a preferred embodiment of the invention, the nucleic acid of thesample which codes for a MIA protein is amplified before testing, e.g.by the well-known PCR technique. A derivatized (labelled) nucleic acidprobe is usually used in the field of nucleic acid diagnostics. Thisprobe is brought into contact with a carrier-bound denatured DNA or RNAfrom the sample and in this process the temperature, ionic strength, pHvalue and other buffer conditions are selected in such a waythat--depending on the length of the nucleic acid sample and theresulting melting temperature of the expected hybrid--the labelled DNAor RNA can bind to homologous DNA or RNA (hybridization, see also J.Mol. Biol. 98 (1975), 503; Proc. Natl. Acad. Sci. USA 76 (1979), 3683).Suitable carriers are membranes or carrier materials based onnitrocellulose (e.g. Schleicher and Schull, BA 85, Amersham Hybond, C.)reinforced or bound nitrocellulose in a powder form or nylon membranesderivatized with various functional groups (e.g. nitro group) (e.g.Schleicher and Schull, Nytran; NEN, Gene Screen; Amersham Hybond M.;Pall Biodyne).

The hybridized DNA or RNA is then detected by incubating the carrier,after thorough washing and saturation to prevent unspecific binding,with an antibody or antibody fragment. The antibody or antibody fragmentis directed towards the substance incorporated into the nucleic acidprobe during the derivatization. The antibody is in turn labelled. Itis, however, also possible to use a directly labelled DNA. Afterincubation with the antibodies, it is washed again in order to onlydetect specifically bound antibody conjugates. The determination is thencarried out via the label of the antibody or antibody fragment accordingto well-known methods.

The detection of the MIA expression can be carried out for example as:

in situ hybridization with immobilized whole cells using immobilizedtissue smears and isolated metaphase chromosomes,

colony hybridization (cells) and plaque hybridization (phages andviruses),

Northern hybridization (RNA detection),

serum analysis (e.g. cell type analysis of cells in serum by slot-blotanalysis),

after amplification (e.g. PCR technique).

The invention therefore includes a method for the detection of nucleicacids which code for a MIA protein which is characterized in that thesample to be examined is incubated with a nucleic acid probe which isselected from the group

a) the DNA sequences shown in SEQ ID NO 1 and 3 or a complementarysequence to these

b) nucleic acids which hybridize with one of the sequences from a),

the nucleic acid probe is incubated with the nucleic acid from thesample and the hybridization of the nucleic acid in the sample andnucleic acid probe is detected, if desired, via a further bindingpartner.

Thus, MIA is a valuable prognostic marker in tumour diagnostics(metastasis, progress).

The invention is elucidated in more detail by the sequence protocols inconjunction with the following examples and figures. In this case

SEQ ID NO: 1--denotes cDNA of human MIA with pre sequence

SEQ ID NO: 2--denotes protein

SEQ ID NO: 3--denotes genomic DNA of MIA.

SEQ ID NO: 4--denotes cDNA of murine MIA with pre sequence

SEQ ID NO: 5--denotes protein

SEQ ID NO: 6--denotes primer

SEQ ID NO: 7--denotes primer

SEQ ID NO: 8--denotes cloning fragment

SEQ ID NO: 9--denotes primer

SEQ ID NO: 10--denotes primer

SEQ ID NO: 11--denotes adaptor

SEQ ID NO: 12--denotes adaptor

SEQ ID NO: 13--denotes fusion protein

SEQ ID NO: 14--denotes fusion protein

SEQ ID NO: 15--denotes primer

SEQ ID NO: 16--denotes primer

SEQ ID NO: 17--denotes primer

SEQ ID NO: 18--denotes fusion-free MIA for expression in E. coli

SEQ ID NO: 19--denotes primer

SEQ ID NO: 20--denotes primer

SEQ ID NO: 21--denotes primer

SEQ ID NO: 22--denotes primer

SEQ ID NO: 23--denotes polylinker

SEQ ID NO: 24--denotes genomic DNA of MIA (allelic variant)

BRIEF DESCRIPTION OF THE FIGURES

    ______________________________________                                        FIG. 1     depicts the invasion-inhibitory activity of                                   human MIA (inhibition of movement of the                                      cells with and without MIA, in %).                                            B 16 + mMIA: Test using murine MIA                                 FIG. 2A    depict the inhibition of T-cell mediated                           and 2B     cytotoxic activity by MIA, expressed as                                       % lysis of CD4.sup.+  T cells.                                     FIG. 3     depicts the inhibition of the cytotoxic                                       acitivity of LAK-cells by MIA.                                     FIG. 4     depicts the inhibition of the                                                 phytohaemagglutinin-dependent lymphocyte                                      proliferation by MIA (concentration of MIA                                    expressed as ng/ml).                                               FIG. 5     depicts the inhibition of the IL-2                                            stimulated PBMC proliferation by MIA                                          (concentration of MIA expressed as ng/ml).                         FIG. 6     shows a plasmid chart of the expression                                       plasmid pQE40-MIA (Example 5a).                                    FIG. 7     shows a plasmid chart of the vector                                           pCMX-PL1 (Example 7a).                                             ______________________________________                                    

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Isolation of the melanoma-inhibiting protein from HTZ 19-dM cells.

HTZ 19-dM cells are cultured as a monolayer in defined serum-free tissueculture medium (50% Dulbecco's minimal essential medium, 50% F-12,Boehringer Mannheim GmbH) containing 0.8 mmol/l L-glutamine (Gibco,U.K.), non-essential amino acids (Gibco, U.K.), 10 μg/ml transferrin(Boehringer Mannheim GmbH, Catalogue No. 1073974), 30 nmol/l sodiumselenite (Sigma) and 4 μg/ml Gentamicin (Merck). The cell culturesupernatant of this culture is removed at intervals of 3 to 4 days ineach case and stored at -70° C. until purification.

For purification, the cell culture supernatants are filtered through a0.45 μm filter (Becton Dickinson, Heidelberg) and concentrated bymembrane ultrafiltration using Amicon YM 2 membranes (exclusion limit2000 D, Amico Danvers Mass., USA) to a final volume of 1% of the initialvolume. The material obtained is dialysed for 30 hours against 0.1 mol/lacetic acid (dialysis membrane with an exclusion limit of 1000 D,Reichelt, Heidelberg) and subsequently ultracentrifuged at 100,000 g forone hour at 4° C. The pellet is discarded and the supernatant islyophilized for further processing.

The lyophilized dialysates are taken up in 1 mol/l acetic acid andpurified further by gel permeation chromatography on a Biogel P-10column (Pharmacia, Uppsala, 2.6×100 cm; Biogel P-10, 200-400 mesh,Biorad Laboratories, Richmond, Calif., USA). The gel material isequilibrated with 1 mol/l acetic acid at 22° C. and the dialysates areapplied at a concentration of 130-145 mg in 5 ml 1 mol/l acetic acid. Itis eluted with 1 mol/l acetic acid at a flow rate of 12 ml/hour and theeluate is collected in 4 ml fractions. Three active fraction pools aredefined by determination of the anti-tumour activity (cf. example 5) ofwhich the middle pool which corresponds to a molecular weight of8000-17000 D is purified further by means of reverse HPLC. For thisthese fractions are firstly lyophilized once and then taken up in 0.1%trifluoro-acetic acid (TFA). 100 μl aliquots of the solution obtainedare applied in each case to the reverse phase HPLC (flow rate 0.5ml/min). Fractions of 750 μl in each case are collected. Further dataconcerning the HPLC separation:

Gradient program:

solution A: 0.06 TFA in water

solution B: 0.056% TFA, 80% acetonitrile

2-25% solution B within 5 min

25-50% solution B within 120 min

50-100% solution B within 5 min return to 2% within 5 min

column: minoRPC (Pharmacia)

HPLC gradient mixer, pump and detector: Pharmacia

The eluate is collected in 1.5 ml fractions. Aliquots are lyophilizedand examined for anti-tumour activity as described in example 5.

Ca. 1 μg melanoma-inhibiting protein can be obtained in this manner from5 l culture supernatant.

EXAMPLE 2 EXAMPLE 2a

Cloning the cDNA coding for the human melanoma-inhibiting protein

The MIA amino acid sequences are determined in a sequencer after Asp-Nand trypsin digestion of the purified protein and repurification of thepeptide fragments obtained in this manner. The C-terminal peptidesequence and one located near to the N-terminus were selected as thebasis for the synthesis of two primers. The primers are degenerateoligonucleotides with added restriction enzyme cleavage sites.

    Upstream primer 1 (sense) (UP 1)                           (SEQ ID NO: 6)

5'TGTGAATTCAGTTIA/TG/CIGCIGAT/CCAA/GGAA/GTG 3' EcoRI site

The diagonal stroke (/) denotes that the base at this position islocated either in front of or behind the diagonal stroke. Thisoligonucleotide is a mixture of 32 different molecules thereby coveringalmost all possible codons. G-T mismatches with the target sequence canonly occur at positions 12 and 13 which does not increase the stabilityof the hybrids but does not reduce it either. An EcoRI linker isadditionally attached to the 5' end in order to be able to easilyreclone a possible product by PCR. A further 3 unspecific bases arelocated in front of this at the 5' end in order that the restrictioncleavage site is not quite at the end since restriction enzymes do notcleave very well at this position.

    Downstream primer 1 (antisense) (DP1)                      (SEQ ID NO: 7)

5'TGTGTCGACTGTTCGTAGAAA/GTCCCATCTTA/GTC 3' SalI site

DP 1 corresponds to 8 the C-terminal amino acids, is 8-fold degenerateand contains a Sal I cleavage site.

The primer DP 1 was used in the following mixture for the specific firststrand synthesis:

5 μl 10×PCR buffer

3 μl HTZ-19 total RNA

was mixed and heated for 5 min to 65° C.

The following were added by pipette:

1 μl 100 mM MgCl₂

10 μl 2.5 mM dNTP

0.5 μl placental RNase inhibitor

2 μl DP 1 (1 μg)

1 μl reverse transcriptase.

After 1 h incubation at 37° C. the following were added to the abovemixture:

1 μl UP 1 (1 μg)

5 μl 10×PCR buffer

70.5 μl H₂ O

1 μl Taq polymerase

The amplification was carried out in 30 cycles with the followingprofile:

94° C. 30 sec.

55° C. 30 sec.

72° C. 60 sec.

After the last cycle the mixture was incubated for a further 7 minutesat 72° C. for the complete elongation of all products.

After a phenol/chloroform extraction and EtOH precipitation, the PCRmixture was digested with EcoRI and SalI for 2 hours at 37° C. Afterenzyme activation, it was subsequently separated in a 5% PAA gel and the320 bp fragment was eluted overnight. Half the eluate was ligatedovernight with 100 ng EcoRI/SalI digested pbluescript. It was possibleto pick a recombinant white colony from the bacteria (E. coli DH5a)which were transformed on the next day and plated out on SOB agar platescontaining Amp and X-Gal/IPTG.

After isolation of the plasmid, sequencing of the recloned insert wascarried out using the T-7 Deaza sequencing kit from Pharmacia. The T-3and T-7 primers (Stratagene) were available as primers. The followingpicture emerged from the overlap of the read sequences (primers are inprint): ##STR1## A lambda gt11 cDNA library was available for cloningthe complete cDNA which had been synthesized from the RNA of HTZ-19melanoma cells growing in a defined medium (dM).

A total of 25 plates each with 8000 pfu were plated out, 2nitrocellulose filters were placed on each and they were hybridized withthe purified MIA-PCR insert labelled by nick translation (50 mlhybridization solution, 2×10⁶ cpm/ml). After a two-day autoradio-graphy,several signals were obtained which gave a corresponding hybridizationsignal on both filters. The corresponding phage-plaques were picked outand subjected to a rescreen. Four dilution steps of each isolated plaquewere plated out for this and the plates with 100-300 pfu were used fortwo further rescreens.

After a 50 ml overnight culture, the lambda DNA could be isolated fromthe plaques isolated in this manner, 40 μg thereof was digested withEcoRI and separated in a 5% PAA gel. The insert was eluted and 8 ng wasused for ligation with 100 ng EcorRI-digested dephosphorylated vector(pbluescript, Stratagene). Half the ligation mixtures were used for thetransformation of competent E. coli DH5a which were plated on SOB/Ampplates containing IPTG and X-Gal for the blue/white selection. Therecombinant colonies were picked out, the plasmid DNA was isolated andthe inserts were sequenced. The sequence of the insert with the completecoding sequence is shown in SEQ ID NO:1.

A plasmid obtained in this manner is pbs L7MIA which was deposited atthe "Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH" (DSM)in Braunschweig, Germany on the 14.07.93 (DSM 8420).

All methods used for cloning are described in detail in J. Sambrook, E.F. Fritsch, T. Maniatis (1989), Molecular cloning: a laboratory manual,2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,USA.

EXAMPLE 2b

Cloning the gene coding for the human melanoma-inhibiting protein

A human genomic DNA library in the bacteriophage lambda FIX II (Elgin etal., Strategies 4 (1991) 8-9) which is commercially available fromStratagene (Heidelberg), was plated on nitrocellulose filters accordingto the established methods (Sambrook et al., Molecular Cloning (1989),Cold Spring Harbor Laboratory Press). In order to be used as ahybridization sample, the cDNA from Example 2a coding for human MIA wasradiolabelled and applied according to the established techniques(Sambrook et al., Molecular Cloning (1989), Cold Spring Habor LaboratoryPress). Prehybridization (2 hours) and hybridization (16 hours) wascarried out at 60° C. in 6×SSC, 5×Denhardt's solution, 100 μg/ml salmonsperm DNA and 0.1% SDS. The ³² P-dCTP-labelled sample was added to thehybridization preparation at a concentration of 1×10⁶ cpm/ml.Thereafter, the filters were washed at 60° C. two times for 20 minutesin 2×SSC, 0.1% SDS, then two times for 20 minutes in 1×SSC, 0.1% SDS,and finally, two times for 20 minutes in 0.25×SSC, 0.1% SDS. The filterswere subsequently dried and then exposed to X-ray film for 24 to 48hours. The plaques which, in this method, yielded a positivehybridization signal were isolated and confirmed by rehybridization. Thehuman genomic DNA contained as an insert in these phages wascharacterized by Southern hybridization using MIA cDNA samples. For thispurpose the phage DNA was cleaved with the restriction endonucleaseXbaI, separated on a 0.8% agarose gel, and subsequently transferred tonitrocellulose according to Southern (J. Mol. Biol. 98 (1975) 503). Theso obtained filters were hybridized with the complete MIA cDNA as asample under the above-described conditions, whereby two XbaI fragmentsof a size of about 1.4 kb and about 2.2 kb gave positive signals. Eachof these two fragments was cloned into the plasmid pbluescriptSK- (Shortet al., Nucl. Acids Res. 16 (1988) 7583-7600; Alting-Meese and Short,Nucl. Acids Res. 17 (1989) 9494) being suitable for sequencing andcommercially available from Stratagene (Heidelberg). The entire sequencecoding for human MIA is located in four exons which are shown with theirflanking sequences in SEQ ID NO:3. Since it was not investigated whetherthere are one or even more additional XbaI fragments between the twoXbaI fragments on which the MIA exons are located, it cannot be ruledout that intron 2 is in actual fact much larger.

EXAMPLE 2c

Cloning of the cDNA coding for the murine melanoma-inhibiting protein

A commercially available (Novagene, N.Y.) cDNA library from a 13.5day-old mouse embryo in the vector lambda EXlox (Palazzolo et al., Gene88 (1990) 25-36) was plated as described in Example 2a. As thehybridization sample there was employed, in radiolabelled form, the cDNAfrom Example 2a which codes for human MIA. The hybridization conditionswere identical to those described in Example 2b, except for thetemperatures applied during hybridization and wash, which were 55° C.here. The cDNA inserts present in the plaques so obtained and confirmedby rehybridization were sequenced. The sequence of the insert containingthe complete coding DNA of the murine MIA is shown in SEQ ID NO:4.

EXAMPLE 2d

Cloning of the gene coding for the murine melanoma-inhibiting protein

In this case a murine genomic DNA library (from the liver of an adultBALB/C mouse in the vector EMBL3 (Frischauf et al., J. Mol. Biol. 170(1983) 827), commercially available from Clontech, Palo Alto Calif.) wassearched in a fashion analogous to Example 2b using the murine MIA cDNAfrom Example 2c as a sample. The conditions were identical to thosedescribed in Example 2b. Also the further proceeding was in analogousfashion.

EXAMPLE 3 EXAMPLE 3a

Determination of the antiproliferative effect of the melanoma-inhibitingprotein on tumour cells

In order to determine the antiproliferative effect of themelanoma-inhibiting protein or of the protein fractions obtainedaccording to example 1 on melanoma cells, exponentially growing HTZ19-dM cells are sown out for 24 hours in 96-well microculture plates(Costar, Zurich) each in 100 μl serum-free medium (see example 1) at adensity of 3×10³ cells per well (according to Chambard et al., J. Cell.Physiol. 135 (1988), 101-107). The cells are then incubated with theprotein fraction to be examined for 4-5 days at 37° C./5% CO₂. Afteraddition of 1 μCi ³ H-thymidine (specific activity 23 Ci/mmol, AmershamBuchler, Braunschweig, Germany) to each, the cells are incubated for afurther 8 hours under identical conditions and subsequently the ³H-thymidine incorporation into the cellular DNA is measured after acidprecipitation in the usual manner by means of a liquid scintillationcounter. The activity of the examined protein fraction is expressed as apercentage of the ³ H-thymidine incorporation of these treated cellscompared to the ³ H-thymidine incorporation in untreated control cells.By use of different concentrations of the isolated melanoma-inhibitingprotein it is possible to determine a concentration at which this ³H-thymidine incorporation is inhibited by 50% compared to the untreatedcontrol, (IC 50 value in the following Table 1).

                  TABLE 1                                                         ______________________________________                                        Antiproliferative effect of the melanoma-inhibiting                           protein on various tumour cells                                               Tumour cell line IC 50 (μg/ml).sup.1)                                      ______________________________________                                        a)      melanoma cell lines                                                           HTZ 19-dM    1.2                                                              ATCC HTB 69  3.7                                                              HTZ 320       1.35                                                            HTZ 318      3.5                                                              ATCC CRL 1424                                                                              2.1                                                      b)      neuroblastoma                                                                 lines                                                                         Kelly        80                                                       c)      glioblastoma                                                                  ATCC HTB17   10                                                       d)      astrocytoma                                                                   HTZ 243       5                                                               HTZ 209       5                                                       ______________________________________                                         .sup.1) The protein obtained after the first purification step was used       (after Biogel P10 column, 50-100fold lower activity than after complete       purification).                                                           

EXAMPLE 3b

Determination of the invasion-inhibiting effect of themelanoma-inhibiting protein on tumour cells

In order to determine the invasion-inhibiting effect of MIA a modifiedBoyden Chamber System (Albini et al., Cancer Res. 47 (1987) 3239-3245)is used. The chambers were obtained from the firm Costar (Blind WellChamber No. 441200). For simulation of a basement membrane-like barrierbetween the chemoattractant in the lower chamber and the cells in theupper chamber, 52 μl matrigel (Becton Dickinson Cat. No. 40234) areapplied onto the polycarbonate filter (pore size 8 μm, Costar No.150446). The lower chamber is filled with 210 μl fibroblastconditioned-medium as the chemoattractant. This medium is obtainedaccording to the following procedure: fibroblasts from normal human skinare maintained between the 10th and the 20th passage in DMEM medium(Gibco) for 24 hours without addition of fetal calf serum. The soconditioned medium is applied in undiluted form as a chemoattractant.Into the upper chamber of the Boyden apparatus are applied 2×10⁵ of thetumour cells to be examined, in 800 μl DMEM (Gibco, without fetal calfserum), with and without MIA active ingredient. Human (see Example 3a orExample 8) or animal tumour cells such as, for example, B16 (ATCC CRL6322) can be tested by the described method with respect to theinhibition of their migration behaviour through MIA. When nomelanoma-inhibiting protein is added, about 10% of the tumour cellsmigrate within a period of about 4 hours from the upper chamber into thelower chamber where they stick to the lower side of the Matrigelmembrane. There, they are fixed, dyed and subsequently counted. If thehuman or murine melanoma-inhibiting protein MIA is added to the upperchamber, then cell migration is strongly inhibited. FIG. 1 shows theinhibition values obtained ( cell count in lower chamber, experimentwith MIA!-\ cell count in lower chamber, experiment without MIA!×100%).

EXAMPLE 4

Determination of the immunological activity

EXAMPLE 4a

MIA inhibits T cell-mediated cytotoxic activity

The CD4⁺ T cell line D7.1 specific for MBP peptide 87-106 is able tolyse targets presenting MBP as well as peptide 87-106 in a standard ⁵¹Cr release assay (targets: Daudi cells, R. Martin, U. Utz, J. E.Coligan, J. R. Richert, M. Flerlage, E. Robinson, R. Stone, W. E.Biddison, D. E. MacFarlin, H. F. MacFarland, Diversity in finespecificity and T cell receptor usage of the human CD⁴⁺ cytotoxic T cellresponse specific for the immunodominant myelin basic protein peptide87-106, J. Immunol. (1992), 148 (5), 1359-1366). After addition of MIA(amount used corresponds to ca. 50-100 ng/ml purified MIA), thepeptide-specific cytotoxicity is inhibited by ca. 55% (FIG. 2a) and theMBP-specific cytotoxicity by ca. 50% (FIG. 2b). As expected theinhibition is slightly dependent on the effector-target ratio (E:T)which was set in this case at a very low level of 1:1 or 5:1 and is thushighly specific (FIG. 2).

EXAMPLE 4b

MIA inhibits the cytotoxic activity of lymphokine-activated peripheralblood lymphocytes (LAK cells)

LAK cells are non-clonally expanded, lymphokine-activated peripheralblood lymphocytes, predominantly T lymphocytes (A. A. Rayner, E. A.Grimm, M. T. Lotze, E. W. Chu, S. A. Rosenberg, Lymphokine activatedkiller (LAK) cells: Analysis of factors relevant for immunotherapy ofhuman cancer, Cancer 55 (1985), 1327-1333). They are used in a standardmanner in many immunological therapy approaches for tumour therapy. Inthe experiment shown in this case, LAK cells are examined for theircytotoxicity towards HTZ-19 melanoma cells as targets in amicrocytotoxicity assay. At effector-target ratios of 1:1, 5:1 and 10:1the maximum cytotoxicity (CTX) reaches almost 40%. This is stronglyinhibited after addition of MIA (concentration as in example 4a) and bya maximum of 80% at a low effector-target ratio which is more likely tobe expected locally i.e.--near the tumour itself--(FIG. 3).

EXAMPLE 4c

MIA inhibits IL-2 dependent and phytohaemagglutinin-dependent lymphocyteproliferation

Peripheral blood lymphocytes (PBMC) can be stimulated in standardizedand a classical manner using phytohaemagglutinin (PHA) and interleukin-2(IL-2) (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,W. Strober, Current protocols in immunology, NIH Monograph, J. WileySons, New York, 1992). In this process T cells are stimulated almostexclusively with PHA and IL-2 stimulates predominantly T lymphocytes butalso B cells with an IL-2 receptor. When they are co-incubated with MIAin the stated dose range (protein purity: after the first purificationstep (Biogel P10 column), the activity is about 50-100-fold lower thanafter complete purification) MIA it is possible to achieve a very stronginhibition of the PHA response (FIG. 4). The IL-2 response is inhibitedin a higher dose range (FIG. 5).

EXAMPLE 5

Recombinant expression of MIA as fusion protein in E. coli

EXAMPLE 5a

Construction of expression vectors

For the expression of MIA as a fusion protein with a protein suitable asa carrier in E. coli there may be applied, for instance, thecommercially available vector pQE40 (Cat. No. 33403, DIAGEN GmbH,Dusseldorf). Into this vector a cDNA fragment coding for MIA in matureform is inserted between the restriction sites SphI and HindIII whichare present once each. This type of fragment is produced most simply byPCR amplification according to the known techniques, using the clonedMIA cDNA as a matrix and two suitable primers(5'-GATGCATGCGGTCCTATGCCCAAGCTG-3' (SEQ ID NO: 9) and5'-GATAAGCTTTCACTGGCAGTAGAAATC-3' (SEQ ID NO: 10). The PCR fragmentobtained is cut with SphI and HindIII and ligated into the vector pQE40treated in the same manner. The resulting plasmid expresses a fusionprotein of DHFR (dihydrofolate reductase as carrier) and MIA. In orderto enable free MIA to be cleaved proteolytically from this fusionprotein, a DNA segment coding for the recognition sequence of the IgAprotease (Ser Arg Pro Pro/Ser) is cloned into between DHFR and MIA. Thisis accomplished by opening the expression plasmid with BgIII (partialrestriction with subsequent isolation of the linearized vector) andSphI, followed by insertion of an adaptor(5'-GATCTAGCCGGCCGCCCAGCCCGGCATG-3' (SEQ ID NO: 11) and5'-CCGGGCTGGGCGGCCGGCTA-3' (SEQ ID NO: 12), hybridized to doublestrand). The resulting expression plasmid pQE40-MIA codes for a fusionprotein of DHFR and MIA, with a cleavage site for IgA protease beinglocated between DHFR and MIA. The fusion protein carries at theN-terminus 6 histidines which can be used for purifying the fusionprotein with the aid of Ni-chelate gel materials. Processes of this kindare described in EP-A 0 282 042 and EP-A 0 253 303 which areincorporated herein by reference. FIG. 6 shows the expression plasmidpQE40-MIA.

The content of MIA can be optimized by eliminating the carrier proteinDHFR and replacing it by a peptide being as small as possible andfulfilling the same functions. Such suitable peptides are, for instance,MetArgGlySerHisHisHisHisHisHisGlySerSerArgProPro (SEQ ID NO: 13) (thispeptide can be cleaved from the immediately following amino acidsequence of the mature MIA by IgA protease; processes of this kind aredescribed in WO 91/11520 which is incorporated herein by reference) orMetArgGlySerHisHisHisHisHisHisGlySerValAspAspAspAspLys- (SEQ ID NO: 14)(this peptide can be cleaved from the immediately following amino acidsequence of the nature MIA by enterokinase). Expression plasmids whichcode for such MIA peptide fusions can be prepared by the followingprocedure: PCR amplification using MIA cDNA (SEQ ID NO:1) as a matrixand the primers 5'-AAAAAGGATCCAGCCGGCCGCCCGGTCCTATGCCCAAGCTGGC-3' (SEQID NO: 15) and 5'-GGCGAGCAGCCAGATCTCCATAG-3' (SEQ ID NO: 16) yields afragment which is recut with BamHI and BgIII. The expression vectorpQE40-MIA is also restricted with BamHI and BgIII; the smaller one ofthe resulting fragments is discarded and replaced by the described PCRfragment. Thereby an expression vector is obtained which, afterinduction, expresses the fusion proteinMetArgGlySerHisHisHisHisHisHisGlySerSerArgProPro-MIA (SEQ ID NO: 13).The fusion proteinMetArgGlySerHisHisHisHisHisHisGlySerValAspAspAspAspLys-MIA (SEQ ID NO:14) is obtained in an absolutely analogous manner using the primers5'-AAAAAAGGATCCGTTGATGATGACGATAAAGGTCCTATGCCCAAGCTGGC-3' (SEQ ID NO: 17)and 5'-GGCGAGCAGCCAGATCTCCATAG-3' (SEQ ID NO: 16).

Additional similar fusion proteins of peptides and MIA can be preparedin an analogous manner and cloned under the control of suitable(preferentially strong and inducible promoters) into one of the numerousplasmids described for E. coli, and brought to expression. Fusion of MIAwith a peptide which, in E. coli, leads to secretion of the fusionprotein into periplasma, followed by cleavage and release of MIA, isanother alternative. This process is described in WO 88/09373 which isincorporated herein by reference.

EXAMPLE 5b

Expression of fusion proteins and recovery of MIA

The expression plasmid pQE40-MIA (or a similar expression plasmid whichcan be obtained, for example, by using another basic vector or anothercarrier protein or carrier peptide; such alternatives are also describedin, besides in Example 5a, Methods of Enzymology 185 (Gene ExpressionTechnology), ed. David V. Goeddel, Academic Press 1991) is transfectedinto a suitable E. coli strain which has a sufficient expression of thelac repressor, so that an inducible expression of the MIA fusion proteincan be achieved. For this purpose there is suitable, for example, thestrain E. coli M15 pREP4! which is commercially available together withpQE40 (Diagen GmbH, Dusseldorf), or other E. coli strains such as, forinstance, UT5600 (Earhart et al., FEMS Microbiology Letters 6 (1979)277-280), or E. coli BL21 (Grodberg and Dunn, J. Bacteriol. 170 (1988)1245-1253), which have been transfected with a lac repressor-expressinghelper plasmid such as, for instance, pUBS520 (described in Brinckmannet al., Gene 85 (1989) 109-114 or in EP-B 0 373 365) beforehand. Next,MIA can be obtained by the following procedure: E. coli M15pREP4/pQE40-MIA! is cultured on LB medium until an optical density of0.6 (measured at 550 nm) is achieved, then IPTG(isopropyl-β-D-thiolgalactopyranoside, Boehringer Mannheim GmbH) isadded at a final concentration of 1 mM and subsequently cultured furtherfor 4 hours. The cells are separated by centrifugation, placed in 100 mMsodium-phosphate buffer at pH 7.5 with 300 mM NaCl and lysed by beingfrozen and thawn three times and subsequently subjected to ultrasonictreatment. To the lysate clarified by centrifugation Ni-NTA-agarose(Diagen GmbH) is added, taking into account the maximum binding capacityas stated by the manufacturer, and incubated overnight at ambienttemperature, while mixing. The gel material so loaded with fusionprotein is separated by low-speed centrifugation, washed two times with100 mM sodium phosphate buffer at pH 7.5 and two times with sodiumphosphate buffer at pH 6.1. Thereafter, MIA is cleaved from the fusionprotein by incubation of the gel material with IgA protease (BoehringerMannheim GmbH) in 100 mM sodium phosphate buffer pH 7.5 overnight at 37°C. The gel material is separated by centrifugation overnight and theMIA-containing supernatant is employed, after sterile filtration, in theactivity tests described in Examples 3 and 4.

EXAMPLE 6 Recombinant expression of fusion-free MIA in E. coli

The DNA sequence coding for MIA is modified in such fashion as to allowfor efficient expression in E. coli. For this purpose PCR amplificationis carried out using human MIA cDNA (SEQ ID NO:1) as a matrix and theprimers 1(5'-AAAAACATATGGGACCAATGCCAAAATTAGCAGATCGTAAATTATGTGCAGATCAGGAG-3' (SEQID NO: 19)) and 2 (5'AAAAAAAGCTTTCACTGGCAGTAGAAATC-3' (SEQ ID NO: 20)).Primer 1 changes the MIA-coding sequence in the N terminal region insuch fashion that, while the MIA amino acid sequence is not changed, theDNA sequence and, thus, the mRNA sequence for E. coli are optimized. Thestart codon Met is added, and at this very site a recognition sequenceof the restriction endonuclease Ndel is inserted, which sequence allowsfor subsequent cloning of the so modified MIA-coding fragment into avector containing a (preferentially strong and inducible) promoter and atranslation initiation sequence (Shine Dalgarno sequence) with asubsequently placed Ndel cleavage site. Primer 2 acts as a3'-counter-primer and contains a HindIII cleavage site, so as to allowfor the insertion of the modified MIA-coding fragment into the vector asan Ndel HindIII fragment. The so modified MIA-coding sequence is shownin SEQ ID NO: 18. An expression plasmid prepared in this manner isp11379 (DSM 9267) which was deposited at "Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH", D-38124 Braunschweig on Jun. 29,1994.

For expression, an expression plasmid for fusion-free MIA is transfectedinto a suitable E. coli strain. Such strains are, in the case of the useof an expression plasmid under the control of lac repressor such as theexpression plasmid p11379, strains which possess a sufficiently highintracellular concentration of lac repressor. These kinds of strains canbe prepared by transfection of a second plasmid such as pREP4 (DiagenGmbH), pUBS 500 or pUBS520 (Brinckmann et al., Gene 85 (1989) 109-114).The applied E. coli strains should preferably have a low proteaseactivity of the cells proper, as is the case, for instance, with E. coliUT5600 (Earhart et al., FEMS Microbiology Letters 6 (1979) 277-280), E.coli BL21 (Grodberg and Dunn, J. Bacteriol. 170 (1988) 1245-1253) or E.coli B. Then, expression cultivation is accomplished in a fashionanalogous to Example 5b. In order to recover MIA, the MIA obtained as aprotein aggregate from E. coli is processed according to the proceduresdescribed in EP 0 241 022, EP 0 364 926, EP 0 219 874 and DE-A 40 37196.

In detail, for example the following procedure is applied for thispurpose: MIA-containing protein aggregates from E. coli fermentations(socalled "inclusion bodies") are solubilized in 6M guanidiniumhydrochloride, 100 mM TrisHCl at pH 8, 1 mM EDTA, subsequently adjustedto a pH of 3 to 4 and dialyzed against 4M guanidinium hydrochloride atpH 3.5. The renaturing of the solubilized protein is then carried out in1M arginine at pH 8, 1 mM EDTA, 5 mM GSH (glutathione, reduced) and 0.5mM GSSG (glutathione, oxidized). From the renaturing preparation, MIAcan be obtained, for instance, after addition of 1.4M ammonium sulfateby adsorption to hydrophobic gel matrices such as Fractogel TSK Butyl(E. Merck, Darmstadt) and subsequent elution in 20 mM TrisHCl at pH 7.

EXAMPLE 7

Recombinant expression of MIA in eukaryotic cells

EXAMPLE 7a

Recombinant expression of MIA in mammalian cells

For this, the human (SEQ ID NO:1) or murine (SEQ ID NO:4) MIA cDNA orthe corresponding genomic DNA segments are ligated into a vector inwhich they are transcribed into mammalian cells, on the basis of astrong promoter-enhancer system (in the case of the genomic MIAfragments, this step is needed because the promoters of MIA proper onlyare active in certain cell types, for example melanomas, and aretherefore not suitable for a general recombinant expression; however,expression can also be accomplished by homologous recombination in vitroas described in example 9). Such promoters and enhancers are mostly fromviruses such as SV40, hCMV, polyoma or retroviruses. As an alternativethere can also be applied promoter-enhancer systems which are specificto a certain cell type or tissue type, such as, for instance, WAP-,MMTV- or immune globuline promoter, or systems which are inducible, suchas, for instance, metallothioneine promoter. This kind of vectorsupplements the MIA cDNA (if the latter is used) with donor and acceptorsignals for RNA processing as well as a signal for poly-A-addition. Forexample, pCMX-pL1 (Umesono et al., Cell 65 (1991) 1255-1266) which isshown in FIG. 7 is such a suitable vector. Into the one and only EcoRIcleavage site of this vector the MIA cDNA provided with EcoRI linkers isligated, wherein it is ensured by restriction analysis with the aid ofthe other cleavage sites in the polylinker of this vector (see SEQ IDNO: 23) that the MIA cDNA is oriented in reading direction of the CMVpromoter. An absolutely analogous procedure is applied when cloning intoother vectors, e.g. into pCDNA3 (Invitrogen, San Diego/USA) or pSG5(Stratagene, LaJolla/USA). The DNA of the so obtained expressionplasmids is prepared from E. coli and transfected into the mammaliancells, applying techniques that are specific to the cell types in theparticular case (Methods of Enzymology 185 (Gene Expression Technology),ed. David V. Goeddel, Academic Press 1991, section V). The expressionplasmid pCMX-pL1-MIA is transfected into the human teratocarcinoma linePA-1sc9177 (Buttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583)according to methods that have been described (Buttner et al., Mol.Cell. Biol. 13 (1993) 4174-4185), wherein 200,000 cells per 100 mmculture dish are transfected with 5 μg DNA. After transfection, thecells are cultured in MEM (Gibco) without addition of fetal calf serum,whereby MIA is detectible in the cell culture supernatant after 48hours.

EXAMPLE 7b

Recombinant expression of MIA in insect cells

For expression in insect cells, a DNA segment coding for MIA, preferablythe human MIA cDNA (SEQ ID NO:1), is inserted into vectors derived fromAcMNPV (Autographa californica nuclear polyhedrosis virus) or BmNPV(Bombyx mori nuclear polyhedrosis virus). For this purpose the MIA cDNAis first of all brought under the control of a strong promoter which issuitable for insect cells (D. R. O'Reilly, L. K. Miller and V. A.Luckow, Baculovirus Expression Vectors--A Laboratory Manual (1992), W.H. Freeman & Co., New York), such as the poIH promoter or the p10promoter. In order to express MIA with the help of the poIH promoter thefollowing procedure is applied: A DNA fragment coding for MIA and havingcleavage sites for the restriction endonucleases EcoRI (adjacent to the5' end of the later MIA transcript) and PstI (adjacent to the 3' end ofthe later MIA transcript) is obtained by PCR amplification according toknown techniques and with the use of the MIA cDNA as a matrix and theprimers 5'-CGTGAATTCAACATGGCCCGGTCCCTGGTGTGC-3' (SEQ ID NO: 21) and5'-TATCTGCAGTCACTGGCAGTAGAAATCCCA-3' (SEQ ID NO: 22). This fragment isrecut with EcoRI and PstI (in order to generate the correspondingcohesive ends) and ligated into the transfer vector pVL 1393 which hasbeen restricted with the same endonucleases (D. R. O'Reilly, L. K.Miller and V. A. Luckow, Baculovirus Expression Vectors--A LaboratoryManual (1992), W. H. Freeman & Co., New York) and is commerciallyavailable (PharMingen, San Diego, Calif., or Invitrogen Corporation, SanDiego, Calif.). The resulting transfer expression plasmid pVL 1393-MIA,for proliferation, is transfected into E. coli K12 and plasmid DNA isprepared according to the established methods. The transfer of the MIADNA, which is under the control of the poIH promoter, from the transferplasmid into the baculovirus vector is accomplished by homologousrecombination according to the established methods (O'Reilly et al.(1992), see supra). For this purpose, 0.5 μg Baculo Gold DNA (linearizedAcNPV virus DNA with lethal deletion and lacZ expression controlled bythe poIH promoter, commercially available from PharMingen, Order No.21100D) and 2 μg pVL13-93-MIA are mixed, incubated at ambienttemperature for 5 minutes and subsequently mixed with 1 ml 125 mM Hepesat pH 7.1, 125 mM CaCl₂, 140 mM NaCl. This mixture is added to 2×10⁶ SF9insect cells (Invitrogen, Order No. B825-01) in a culture dish of adiameter of 60 mm which was coated with 1 ml Grace's Medium with 10%fetal calf serum beforehand. After 4 hours of incubation at 4° C. theDNA-containing medium is removed and the cells are incubated in freshmedium at 27° C. for a period of 4 days. The recombinant baculovirusesobtained in this manner are subsequently purified two times via plaqueformation (O'Reilly et al. (1992), see supra), wherein viruses whichhave inserted MIA by homologous recombination are distinguished from theemployed wild type viruses (AcNPV with lethal deletion and lacZexpression controlled by the poIH promoter, commercially available fromPharMingen, Order No. 21100D) by the absence of β-galactosidase activity(optically recognizable by the absence of blue colour in the presence of5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). With a so obtainedMIA-expressing recombinant baculovirus SF9 cells are infected (MOI=20pfu/cell) according to the established methods (O'Reilly et al. (1992),see supra) and incubated further for at least 36 hours at 27° C. inserum-free medium (Cell/Perfect Bac serum free insect cell culturemedium, Stratagene, Order No. 205120). Subsequently the cell culturesupernatants are removed, viruses contained in the supernatant areseparated by ultracentrifugation (Beckmann Ti 60 Rotor, 30,000 rpm) andthereafter the supernatant is filtered through a Microcon 100 Filter(Amicon, exclusion limit 100 kD). The so obtained MIA-containingsolution can be used in the tests described in Examples 3 and 4, or canbe purified further in accordance with Example 1.

EXAMPLE 8

Detection of MIA mRNA in various cells

The detection of expression of MIA in a particular cell and, thus, ofthe presence of MIA mRNA, can be accomplished, on the one hand, with theestablished methods of nucleic acid hybridization such as, for instance,Northern hybridization, in situ hybridization, dot or slothybridization, and diagnostic techniques derived therefrom (Sambrook etal., Molecular Cloning--A Laboratory Manual (1989), Cold Spring HarborLaboratory Press; Nucleid Acid Hybridisation--A Practical Approach(1985), eds. B. D. Hames and S. J. Higgins, IRL Press; WO 89/06698, EP-A0 200 362, U.S. Pat. No. 2,915,082, EP-A 0 063 879, EP-A 0 173 251, EP-A0 128 018). On the other hand, there may be applied methods from thelarge variety of amplification techniques, using MIA specific primers(PCR Protocols--A Guide to Methods and Applications (1990), eds. M. A.Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Academic Press Inc;PCR--A Practical Approach (1991), eds. M. J. McPherson, P. Quirke, G. R.Taylor (1991), IRL Press). Tables 2A and 2B show MIA expression invarious human tumours, tumour lines and normal cells, which wasdetermined in this case by Northern hybridization using theradiolabelled human MIA cDNA (SEQ ID NO:1). For this purpose the RNA wasisolated from the listed cells according to the method of Chomczynskiand Sacchi, Anal. Biochem. 162 (1987) 156-159. 20 μg total RNA wereseparated on a 1% agarose formaldehyde gel and transferred to nylonmembranes (Amersham, Braunschweig) according to standard methods(Sambrook et al., Molecular Cloning--A Laboratory Manual (1989), ColdSpring Harbor Laboratory Press). As a sample, the complete human MIAcDNA (SEQ ID NO:1) was radiolabelled (Feinberg and Vogelstein, Anal.Biochem. 137 (1984) 266-267). Hybridization was carried out at 68° C. in5×SSC, 5×Denhardt's, 0.5% SDS, 10% dextran sulfate and 100 μg/ml salmonsperm DNA. Thereafter the membranes were washed two times per hour in1×SSC at 68° C. and then exposed to X-ray film.

                  TABLE 2A                                                        ______________________________________                                                                 Positive at                                          Tumour        Number tested                                                                            Northern Blotting                                    ______________________________________                                        Astrocytoma   10         3                                                    Oligodendroglioma                                                                           4          0                                                    Ependymoma    3          0                                                    Neuroblastoma 4          0                                                    Glioblastoma  13         0                                                    Colon CA      2          1                                                    Malignant     6          6                                                    melanoma                                                                      CNS metastasis                                                                Medulloblastoma                                                                             2          0                                                    carcinoma of the                                                                            1          0                                                    breast                                                                        bronchial     2          0                                                    carcinoma                                                                     CNS metastasis                                                                ______________________________________                                    

                  TABLE 2B                                                        ______________________________________                                                                 Positive at                                          Normal cells  Number tested                                                                            Northern Blotting                                    ______________________________________                                        Embryonic fibro-                                                                            2          0                                                    blasts                                                                        mononucleic blood                                                                           3          0                                                    cells (3 donours)                                                             ______________________________________                                    

EXAMPLE 9

Use of MIA coding nucleic acids for therapeutic purposes

MIA inhibits the proliferation and metastasising of tumour cells. In ananimal model or a patient this effect may be caused not only byexogenous introduction of MIA protein but also by insertion of a DNAsegment which either codes for MIA under a suitable promoter or containsa suitable promoter capable of integrating before the cell's own MIAgene into the genome by homologous recombination. In the latter case,this promoter must be flanked by sequence portions which are to thehighest possible extent homologous, or preferably even identical, to thesequences of the human (or, in the case of an animal model, animal) MIAgene in the 5'-untranslated region (see e.g. WO 91/09955). By thisprocess it is possible to achieve that the tumour cell is expressing MIAto an increased extent and, thus, inhibits its own proliferation andmetastasising if the DNA segments are inserted in the tumour cellproper. In many cases, however, the corresponding gene segment will notneed to be inserted specifically and exclusively into the tumour cellsproper, because also an expression in other body cells, preferablyadjacent to the tumour, will bring about inhibition of the tumour cellsthrough increased MIA release. The following example shows thetherapeutic effect of a MIA-coding DNA segment in an animal model.

The injection of murine B16 melanoma cells (ATCC CRL 6322) into thecaudal vene of C57BL mice, followed by quantification of lungmetastases, is an established in vivo model of metastasis formation.100,000 cells of the melanoma line B16 were injected behind the eyeballsof C57BL mice (day 1: 16 animals). After 48 hours, in 8 animals, 100 μgof the MIA expression plasmid pCMX-PL1-MIA (Example 7a) in TE (10 mMTrisCl pH 8.0, 1 mM EDTA), mixed with DOTAP transfection reagent(Leventis and Silvius, Biochim. Biophys. Acta 1023 (1990) 124-132,commercially available from Boehringer Mannheim GmbH, Cat. No. 1202375),were injected in each case into the caudal vene. The control group (8animals) was given the same plasmid, however without MIA sequences.After 13 days, 6 of 8 animals from the control group without MIA haddeveloped a local tumour; the average number of metastases in the lung,spleen, kidney and liver was 7.8. In the group which had been given theMIA coding plasmid, only 4 of 8 animals developed a local tumour and theaverage number of metastases was 2.7.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 459 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 40..432                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 40..111                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 112..432                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CCAGCACCCCCTTGCTCACTCTCTTGCTCACAGTCCACGATGGCCCGGTCCCTG54                      MetAlaArgSerLeu                                                               24-20                                                                         GTGTGCCTTGGTGTCATCATCTTGCTGTCTGCCTTCTCCGGACCTGGT102                           ValCysLeuGlyValIleIleLeuLeuSerAlaPheSerGlyProGly                              15-10-5                                                                       GTCAGGGGTGGTCCTATGCCCAAGCTGGCTGACCGGAAGCTGTGTGCG150                           ValArgGlyGlyProMetProLysLeuAlaAspArgLysLeuCysAla                              1510                                                                          GACCAGGAGTGCAGCCACCCTATCTCCATGGCTGTGGCCCTTCAGGAC198                           AspGlnGluCysSerHisProIleSerMetAlaValAlaLeuGlnAsp                              152025                                                                        TACATGGCCCCCGACTGCCGATTCCTGACCATTCACCGGGGCCAAGTG246                           TyrMetAlaProAspCysArgPheLeuThrIleHisArgGlyGlnVal                              30354045                                                                      GTGTATGTCTTCTCCAAGCTGAAGGGCCGTGGGCGGCTCTTCTGGGGA294                           ValTyrValPheSerLysLeuLysGlyArgGlyArgLeuPheTrpGly                              505560                                                                        GGCAGCGTTCAGGGAGATTACTATGGAGATCTGGCTGCTCGCCTGGGC342                           GlySerValGlnGlyAspTyrTyrGlyAspLeuAlaAlaArgLeuGly                              657075                                                                        TATTTCCCCAGTAGCATTGTCCGAGAGGACCAGACCCTGAAACCTGGC390                           TyrPheProSerSerIleValArgGluAspGlnThrLeuLysProGly                              808590                                                                        AAAGTCGATGTGAAGACAGACAAATGGGATTTCTACTGCCAG432                                 LysValAspValLysThrAspLysTrpAspPheTyrCysGln                                    95100105                                                                      TGAGCTCAGCCTACCGCTGGCCCTGCC459                                                (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 131 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetAlaArgSerLeuValCysLeuGlyValIleIleLeuLeuSerAla                              24- 20-15-10                                                                  PheSerGlyProGlyValArgGlyGlyProMetProLysLeuAlaAsp                              515                                                                           ArgLysLeuCysAlaAspGlnGluCysSerHisProIleSerMetAla                              101520                                                                        ValAlaLeuGlnAspTyrMetAlaProAspCysArgPheLeuThrIle                              25303540                                                                      HisArgGlyGlnValValTyrValPheSerLysLeuLysGlyArgGly                              455055                                                                        ArgLeuPheTrpGlyGlySerValGlnGlyAspTyrTyrGlyAspLeu                              606570                                                                        AlaAlaArgLeuGlyTyrPheProSerSerIleValArgGluAspGln                              758085                                                                        ThrLeuLysProGlyLysValAspValLysThrAspLysTrpAspPhe                              9095100                                                                       TyrCysGln                                                                     105                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3565 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 1378..1449                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1378..1504                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1586..1719                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 2804..2914                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 3232..3252                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: -                                                               (B) LOCATION: one-of(2216)                                                    (D) OTHER INFORMATION: /note= "N in position 2216                             denotes an indefinite number ans sequence of                                  nucleotides"                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TCTAGACANATAAAAATAAAAGAAATCATCCAAGAATGGTGACTTGCCTACTATTCTACT60                CGAGAGGCTGAGAGGGGAGGATTTCTTGACCCCGGNAGTTTAAGGATGCAGTGAGCTATG120               ATCACATCACTGTACTTCAGCCTGAGCAACAGCAAGATCCTGTCTCTAAAAATTAAATAA180               GGCTGGGCTTGGTGGCTCATGCTGTAATCCCAGCACTTTGGAAGGCCATGGTGGGCAGAT240               TGCTTGAGCCCAGGAGTTTGAGACGAGGCTGGGCAACATGACGAAACCCCGGCTCTACCA300               AAAAATACAAAAAATTAACTGGGCATAATGGTACATGTCTGTGGTCCCAGCTACTCGGTA360               GGCTGAGGTGGGAGGAATGCTTGAGCCCAGGAAATAGGGGCTACAGTGAACCAGGATGAT420               GCCAGTGCACTCCAACCTGGGCAACAGAGCAAGACTCTACCTCAAAATAATTTAAAAAAA480               TGGATTAATTGGGCATAGGTGGCTTGTGCCTGTAGTCCCAGTTACTCAGGAGCCTGAGGT540               GGGAGGATTGCCTGAGTCTAGGAGGTTGAGGCTGCAGTGAGCCGGGATGGCACCATTGCA600               CTCCACCTGGGCAACAGGGTGAGACCCTGTCTCAAAAAAGAAAAAAAAGGGAGGGGTTAT660               AATCACTCCTCCTGACATGATACAGAGTATCCATTTGAGTTCATAACATAAATATGTACT720               TGGTGAATGCTCTGTAACTATTGGTGAATGCTCTGTAACTATTGGCTTTTTTATTGTTCC780               CATTTTACATATAAGGAAGCTGAGGCTTTGTGAGGAGAAATAGCTTAGCCCAGGTCATCC840               AGTGGGAAGCGTCTGGTGCAGAGGAATAGTGATCAGGGTGGGACTTTGCCTAGCCTAAGG900               TTCAGCATACAATATTCAGTCAGTACTCAAGGGCTGGGCTGTTTCTGGTAATCAAAGGGC960               CTGCCTTGTCCTCCTCCCCCACAGCAGGAAATTCCAAGGTGGTTTTCTTTACAGGCTCCT1020              CCGCTTCTGTGGCCAGAGGGGACAGCGGAGGACCCCAGGTACCTAAGCCAACTCAAGAGA1080              AGATGGAATTGAATATTTCAACCACCTTATCTAGGCCTCTGTGATTGTTGAGGAGGGGGC1140              TGTCACTGGGAAAGTTGTGAGCTGCTTTGGACCTTATCTGGGAATTTCCTTGGGCCTTAC1200              AGCTTTACCCTATCCTTGAAATGGTTCTGGTTTCATAGCAACTTCTAGGTGGTGTGGGCG1260              AAGTTTGGGACTGGTTTAGGGCGGGGACAAGACCAAGAACACAAGTTTCCTTGTACGGGA1320              GAGAGGAAATTGGAGACCCCAGCACCCCCTTGCTCACTCTCTTGCTCACAGTCCACGATG1380              GCCCGGTCCCTGGTGTGCCTTGGTGTCATCATCTTGCTGTCTGCCTTCTCCGGACCTGGT1440              GTCAGGGGTGGTCCTATGCCCAAGCTGGCTGACCGGAAGCTGTGTGCGGACCAGGAGTGC1500              AGCCGTAAGAATGGGGAGGGGTAGAATTGGGCTTGGGTGTTAGCCTGTGTGGATGTGCTG1560              CATTCCCCTTCTATTCCTTCCCTAGACCCTATCTCCATGGCTGTGGCCCTTCAGGACTAC1620              ATGGCCCCCGACTGCCGATTCCTGACCATTCACCGGGGCCAAGTGGTGTATGTCTTCTCC1680              AAGCTGAAGGGCCGTGGGCGGCTCTTCTGGGGAGGCAGCGTGCGTCTTGGGAGAGTGAAA1740              GAGGGAAGGGTACAGAGCTGGGGTAGACTCATTATCCCCATGAAGGGAAGATTTGAGGGG1800              GGTGAACTGAAATAGACATTGTGGGGGGATATTGTTACTTACTTTATTTTATTTGCTTAT1860              TATTTTTTAATTTTTTCCGAGACAGAGTCTTGCTCTGTCACCCAGGCTGGATGCAATGGC1920              ACGATCTCGGCTCACTGTAACCTCCACCTCTTGGGTTTAAGCGATTCTCCAGCCTCAGCC1980              TCCCAAGTACCTGGGATTACAGGCATGCACCACCACACCTNNTAATTTTTGTATTTTTAG2040              TAGAGACAGGGTTTTACCATATTGGCCAGGCTGGTCTTGAACTCCTGACCTCATGATCTG2100              CCCGCCTTGGCTCCCGGAGTGCTGGGATTACAGGTGTGAGCCACTGGCCCCCCAGCCTAT2160              TTTCACTTTATTTACCAATTTTAGGACCTGATATGGTCCCANNNTCTGTTCTAGANTCTA2220              GACACCAAGATACAACAACAAATGATCCTTTTTATTCTAATGGAGGGAAATGAACAAAAA2280              GCAAGGCATAAAAAATAGCAGCAGCCGGGCACAGTAGCTCACACCTGTAATCCCAAGTAA2340              GGCCAAGTNNGGAGGATAGCTTGAGCCCAGGAGTTCGAGACCAGCCTGGGCAACATAGCA2400              AGACCCCCATCTCTATAAAAAAAAATTTAAAATTAACTGGGCATCATGGCATGTGTCTGT2460              GGTCCCGGCTACTCGGGAGGCTGAGGTGGGAGGATTGCTTGATCCCAGAAGTTGAGGCTG2520              CAGTGAGCCGTGATCATGCTACTGCACCTCAACCTGGCCGACACAATGAGACCCTGTTTC2580              CAAAATAATAATAATAAAAGCAAATATGCGCTGCTGTGAGAATTAACAGAGACTTACTTG2640              GGTGTTCAGAAAGGGCCTCTGAACAGGTGGCATTTAAGCTGAGATTCATATGACAAGGAT2700              GGAGCAGTTATGTGGAGATCAGGGAGAGGGGAGAATGCAAAGGCCTTCAGCAGGCACAAG2760              CTTGCCATCTTCCAGACCCTAGCTTTTAACTCCTCTTCCCCAGGTTCAGGGAGATTACTA2820              TGGAGATCTGGCTGCTCGCCTGGGCTATTTCCCCAGTAGCATTGTCCGAGAGGACCAGAC2880              CCTGAAACCTGGCAAAGTCGATGTGAAGACAGACGTGAGTGTCATGGGGGCTGGCAAGAA2940              ATGTGGGGGGAGGACCCTTAGGTTGTGGGGATGGGCAAAAATGCTCCCACACTTGGCTCC3000              CTGGCCGCCTAGGTATGTGCGCTGGGAGAAATTCTTTCCCTGCCTCAATTTTCTCACCAG3060              TAAAATGGGTCCAGTTGGGAGGTGCAAAGATTAGAGGGCTCTAGGCTAATTTGCATAGCA3120              NNTGTGTGGCCAGACCTGGGCCCTGCAGCTGCAGCCTTTGCTAAAACCACTAGATCCTTT3180              GTGGTGTGACCGCTGGTTTTCTTTCCACTGTTTCCCCTTTCTCTTTTTCAGAAATGGGAT3240              TTCTACTGCCAGTGAGCTCAGCCTACCGCTGGCCCTGCCGTTTCCCCTCCTTGGGTTTAT3300              GCAAATACAATCAGCCCAGTGCAAACGGCTCGTCTCCGTGGTCTTTGGGGTGGGGTAGGG3360              TAGGGTGGGGACTGTACAAATGAAATGTTTCTCTAGGTTGCTGAATCTAACCAATTAACC3420              CGCTGCCTGTGGTAACGTCAGTGGTTGCTAGGCAGAGTTTCGCTGATGAAAGCCCTGTGC3480              AGTAGGAGCGCTCCTAAGCTTAGGTTTCGACACAAGCAAAGAAAACCTAAGCAGCCCAAC3540              TAGGGATTGTAGTGTCCTCTCTAGA3565                                                 (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 581 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 110..499                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 110..178                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 179..499                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      AAGCGGCGGAGACAGGATCGAGAACACAGGTTTCCTTGATATTCAGCCTGGAAGGAGGGC60                AGGAGGAGCCCAGAGACCTCGTTCTTCACTTGGTCATTCTCAGTCCATGATGGTG115                    MetVal                                                                        23                                                                            TGGTCCCCAGTGCTCCTTGGCATCGTCGTCTTGTCTGTTTTTTCAGGG163                           TrpSerProValLeuLeuGlyIleValValLeuSerValPheSerGly                              20-15-10                                                                      CCTAGCAGGGCTGATCGAGCTATGCCCAAGCTGGCTGACTGGAAGCTG211                           ProSerArgAlaAspArgAlaMetProLysLeuAlaAspTrpLysLeu                              51510                                                                         TGTGCGGACGAGGAATGCAGCCATCCTATCTCCATGGCTGTGGCCCTC259                           CysAlaAspGluGluCysSerHisProIleSerMetAlaValAlaLeu                              152025                                                                        CAGGACTACGTGGCCCCTGATTGCCGCTTCTTGACTATATATAGGGGC307                           GlnAspTyrValAlaProAspCysArgPheLeuThrIleTyrArgGly                              303540                                                                        CAAGTGGTGTATGTCTTCTCCAAGTTGAAGGGCCGTGGGCGCCTTTTC355                           GlnValValTyrValPheSerLysLeuLysGlyArgGlyArgLeuPhe                              455055                                                                        TGGGGAGGCAGTGTTCAGGGAGGTTACTATGGAGACCTGGCAGCCCGC403                           TrpGlyGlySerValGlnGlyGlyTyrTyrGlyAspLeuAlaAlaArg                              60657075                                                                      CTGGGCTATTTCCCCAGTAGCATTGTCCGGGAGGACCTGAACTCGAAA451                           LeuGlyTyrPheProSerSerIleValArgGluAspLeuAsnSerLys                              808590                                                                        CCTGGCAAAATTGATATGAAGACCGATCAATGGGATTTCTACTGCCAG499                           ProGlyLysIleAspMetLysThrAspGlnTrpAspPheTyrCysGln                              95100105                                                                      TGAGCTCAGCCTACCGCTATCCCTGCAGTTACCTTCCGGCTCTATGCAAATACAGCAGCC559               AATGGCAAAAAAAAAAAAAAAA581                                                     (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      MetValTrpSerProValLeuLeuGlyIleValValLeuSerValPhe                              23-20-15- 10                                                                  SerGlyProSerArgAlaAspArgAlaMetProLysLeuAlaAspTrp                              515                                                                           LysLeuCysAlaAspGluGluCysSerHisProIleSerMetAlaVal                              10152025                                                                      AlaLeuGlnAspTyrValAlaProAspCysArgPheLeuThrIleTyr                              303540                                                                        ArgGlyGlnValValTyrValPheSerLysLeuLysGlyArgGlyArg                              455055                                                                        LeuPheTrpGlyGlySerValGlnGlyGlyTyrTyrGlyAspLeuAla                              606570                                                                        AlaArgLeuGlyTyrPheProSerSerIleValArgGluAspLeuAsn                              758085                                                                        SerLysProGlyLysIleAspMetLysThrAspGlnTrpAspPheTyr                              9095100105                                                                    CysGln                                                                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: -                                                               (B) LOCATION: one-of(14, 17, 20)                                              (D) OTHER INFORMATION: /label=N                                               /note= "N denotes I (inosin)"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      TGTGAATTCAGTTNWSNGCNGAYCARGARTG31                                             (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      TGTGTCGACTGTTCGTAGAARTCCCATCTTRTC33                                           (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 305 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- RNA                                                 (B) LOCATION: join(1..29, 277..305)                                           (D) OTHER INFORMATION: /function="Primer"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GAATTCAAGTTTTCGGCGGATCAGGAGTGCAGCCACCCTATCTCCATGGCTGTGGCCCTT60                CAGGACTACATGGCCCCCGACTGCCGATTCCTGACCATTCACCGGGGCCAAGTGGTGTAT120               GTCTTCTCCAAGCTGAAGGGCCGTGGGCGGCTCTTCTGGGGAGGCAGCGTTCAGGGAGAT180               TACTATGGAGATCTGGTCGCTCGCCTGGGCTATTTCCCCAGTAGCATTGTCCGAGAGGAC240               CAGACCCTGAAACCTGGCAAAGTCGATGTGAAGACAGATAAATGGGATTTCTACGAACAG300               TCGAC305                                                                      (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GATGCATGCGGTCCTATGCCCAAGCTG27                                                 (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GATAAGCTTTCACTGGCAGTAGAAATC27                                                 (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     GATCTAGCCGGCCGCCCAGCCCGGCATG28                                                (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     CCGGGCTGGGCGGCCGGCTA20                                                        (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     MetArgGlySerHisHisHisHisHisHisGlySerSerArgProPro                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     MetArgGlySerHisHisHisHisHisHisGlySerValAspAspAsp                              151015                                                                        AspLys                                                                        (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     AAAAAGGATCCAGCCGGCCGCCCGGTCCTATGCCCAAGCTGGC43                                 (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     GGCGAGCAGCCAGATCTCCATAG23                                                     (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     AAAAAAGGATCCGTTGATGATGACGATAAAGGTCCTATGCCCAAGCTGGC50                          (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 330 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 7..327                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- RNA                                                 (B) LOCATION: 4..6                                                            (D) OTHER INFORMATION: /function="Startcodon Met"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     CATATGGGACCAATGCCAAAATTAGCAGATCGTAAATTATGTGCAGATCAGGAGTGCAGC60                CACCCTATCTCCATGGCTGTGGCCCTTCAGGACTACATGGCCCCCGACTGCCGATTCCTG120               ACCATTCACCGGGGCCAAGTGGTGTATGTCTTCTCCAAGCTGAAGGGCCGTGGGCGGCTC180               TTCTGGGGAGGCAGCGTTCAGGGAGATTACTATGGAGATCTGGCTGCTCGCCTGGGCTAT240               TTCCCCAGTAGCATTGTCCGAGAGGACCAGACCCTGAAACCTGGCAAAGTCGATGTGAAG300               ACAGACAAATGGGATTTCTACTGCCAGTGA330                                             (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     AAAAACATATGGGACCAATGCCAAAATTAGCAGATCGTAAATTATGTGCAGATCAGGAG59                 (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     AAAAAAAGCTTTCACTGGCAGTAGAAATC29                                               (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     CGTGAATTCAACATGGCCCGGTCCCTGGTGTGC33                                           (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     TATCTGCAGTCACTGGCAGTAGAAATCCCA30                                              (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 260 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     GTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTAACTGGC60                TTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGTACCAGATATCAGGATCC120               CCCGGGCTGCAGGAATTCGATATCAAGCTTCTCGAGGGGGGGCCCGGTACCGATCCTGGC180               CAGCTAGCTAGTAGCTAGAGGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAA240               TTGGACAAACTACCTACAGA260                                                       (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 596 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(40..111, 40..166, 214..347, 393..503, 549                  ..569)                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 40..111                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 40..166                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 214..347                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 393..503                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 549..569                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: -                                                               (B) LOCATION: one-of(194, 369, 527)                                           (D) OTHER INFORMATION: /note= "N in positions 194, 369                        and 527 denotes an indefinite number and sequence                             of nucleotides "                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     CCAGCACCCCCTTGCTCACTCTCTTGCTCACAGTCCACGATGGCCCGGTCCCTGGTGTGC60                CTTGGTGTCATCATCTTGCTGTCTGCCTTCTCCGGACCTGGTGTCAGGGGTGGTCCTATG120               CCCAAGCTGGCTGACCGGAAGCTGTGTGCGGACCAGGAGTGCAGCCGTAAGAATGGGGAG180               GGTAGAATTGGGNCCCTTCTATTCCTTCCCTAGACCCTATCTCCATGGCTGTGGCCCTTC240               AGGACTACATGGCCCCCGACTGCCGATTCCTGACCATTCACCGGGGCCAAGTGGTGTATG300               TCTTCTCCAAGCTGAAGGGCCGTGGGCGGCTCTTCTGGGGAGGCAGCGTGGGTCTTGGGA360               GAGTGAAANAGCTTTTAACTCCTCTTCCCCAGGTTCAGGGAGATTACTATGGAGATCTGG420               CTGCTCGCCTGGGCTATTTCCCCAGTAGCATTGTCCGAGAGGACCAGACCCTGAAACCTG480               GCAAAGTCGATGTGAAGACAGACGTGGAGTGTCATGGGGGCTGGCANTTTCCCCTTTCTC540               TTTTTCAGAAATGGGATTTCTACTGCCAGTGAGCTCAGCCTACCGCTGGCCCTGCC596                   __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule, the complementarysequence of which hybridizes to SEQ ID NO: 1 or SEQ ID NO: 3, at 55°-66°C., 6×SSC, 5×Denhardt's solution, 100 ug/ml salmon sperm DNA or 68° C.,5×SSC, 5×Denhardt's solution, 0.5% SDS, 10% dextran sulfate and 100ug/ml salmon sperm DNA.
 2. The isolated nucleic acid molecule of claim1, which encodes a protein having melanoma inhibiting activity, andwhich inhibits growth of cell lines HTZ 19dM and ATCC CRL
 1424. 3. Theisolated nucleic acid molecule of claim 2, which encodes a proteinhaving the amino acid sequence of SEQ ID NO:
 2. 4. The isolated nucleicacid molecule of claim 2, which encodes a protein having an amino acidsequence comprising amino acids 1 through 107 of SEQ ID NO:
 2. 5. Theisolated nucleic acid molecule of claim 2, having the nucleotidesequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:
 4. 6. Arecombinant expression vector comprising the isolated nucleic acidmolecule of claim 2, operably linked to a promoter.
 7. A recombinantcell comprising the isolated nucleic acid molecule of claim
 2. 8. Therecombinant cell of claim 7, wherein said cell is a prokaryotic cell. 9.The recombinant cell of claim 7, wherein said cell is a eukaryotic cell.10. The recombinant cell of claim 8, wherein said prokaryotic cell is anE. coli cell.
 11. The recombinant cell of claim 9, wherein saideukaryotic cell is a mammalian cell.
 12. A process for recombinantproduction of a protein which has melanoma inhibiting activity and whichinhibits growth of cell lines HTZ 19-dM and ATCC-CRL 1424, comprisingtransforming or transfecting a cell with the isolated nucleic acidmolecule of claim 2, to produce said protein and isolating said proteinfrom said cell.
 13. The process of claim 12, wherein said isolatednucleic acid molecule has a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO:
 4. 14. A methodfor detecting a nucleic acid molecule which encodes a protein havingmelanoma inhibiting activity, comprising incubating a sample with theisolated nucleic acid molecule of claim 2 and determining anyhybridization of said isolated nucleic acid molecule to a target nucleicacid molecule as a determination of presence of a nucleic acid moleculewhich encodes a protein having melanoma inhibiting activity.
 15. Themethod of claim 14, further comprising amplifying said target nucleicacid molecule prior to detecting it.
 16. A method for producing aprotein which has melanoma inhibiting activity and which inhibits growthof cell lines HTZ 19-dM and ATCC 1424, comprising:homologouslyrecombining a DNA construct into a genome of a cell which comprises anendogenous gene for said protein, said DNA construct comprising aregulatory element which is capable of stimulating expression of saidendogenous gene when operably linked thereto, and at least one DNAtargeting segment having a nucleotide sequence homologous to a regionwithin or proximal to said endogenous gene, culturing said cell toproduce said protein, and recovering said protein.
 17. A process forproducing a protein which has melanoma inhibiting activity and whichinhibits growth of cell lines HTZ 19-dM and ATCC 1424,comprising:homologously recombining a DNA construct into a genome of amammalian cell, said DNA construct comprising:(a) a nucleic acidmolecule which encodes said protein, (b) a DNA regulatory elementcapable of stimulating expression of (a), and (c) at least one DNAtarget segment homologous to a region of said genome; culturing saidmammalian cells following said homologous recombination protein, andrecovering said protein.
 18. An isolated protein which has melanomainhibiting activity and which inhibits growth of cell lines HTZ 19-dMand ATCC CRL 1424, which is encoded by(a) a nucleic acid molecule havingthe nucleotide sequence set forth in SEQ ID NO: 1, (b) a nucleic acidmolecule having the nucleotide sequence set forth in SEQ ID NO: 3, (c) anucleic acid molecule comprising nucleotides 112-432 of SEQ ID NO: 1,(d) a nucleic acid molecule comprising nucleotides 40-432 of SEQ ID NO:1, (e) a nucleic acid molecule having the nucleotide sequence set forthin SEQ ID NO: 4, (f) a nucleic acid molecule comprising nucleotides112-494 of SEQ ID NO: 4, (g) a nucleic acid molecule comprisingnucleotides 179-499 of SEQ ID NO: 4, (h) a nucleic acid molecule, whichencodes a protein having an amino acid sequence identical to the aminoacid sequence of a protein expressed by any of (a), (b), (c), (d), (e),(f) or (g).
 19. The isolated protein of claim 18, having a molecularweight to about 11 kilodaltons as determined by SDS-PAGE, undernon-reducing conditions.
 20. The isolated protein of claim 19, obtainedfrom cell culture supernatant of cell line HTZ 19dM via gelchromatography and reversed phase, high performance liquidchromatography.
 21. The isolated protein of claim 18, having an aminoacid sequence identical to the amino acid sequence encoded bynucleotides 40-432 or 112-432 of SEQ ID NO:
 1. 22. The isolated proteinof claim 18, having an amino acid sequence identical to the amino acidsequence encoded by nucleotides 110-499 or 179-499 of SEQ ID NO:
 4. 23.Isolated cell line HTZ 19-dM (DSM ATCC 2133).
 24. Antibody whichspecifically binds to the protein of claim 18 obtained by immunizing ananimal with said protein and isolating an antibody produced by saidanimal in response to said protein.
 25. A process for making an antibodywhich binds with the protein of claim 18, comprising immunizing asubject animal with said protein and recovering any antibodies producedin response thereto.
 26. Process for making a therapeutic agent usefulin tumor therapy comprising combining the protein of claim 18 with apharmaceutical auxiliary substance, a filler, or an additive. 27.Therapeutic composition comprising the isolated protein of claim 18 anda pharmaceutical auxiliary substance, a filler, or an additive.